1. Field of the Invention
There are provided oligomers of 3'-deoxyadenosine-5'-triphosphate and the terminally dephosphorylated analogs thereof which are useful anti-viral agents and methods for producing said materials.
2. Discussion of the Relevant Art
The full nomenclature of the subject matter of the present invention involves extremely long terms. It is customary for those skilled in the art to abbreviate these terms in a manner well known to them. These general and customary abbreviations are set forth herein below and will be utilized in the text of this specification.
Abbreviations: EBV, Epstein Barr virus; (2'-5')(A).sub.n synthetase, ATP: (2'-5')oligo(A) adenyltransferase (EC 2.7.7.-); (2'-5')pppA(pA).sub.n or (2'-5')oligo(A), oligomer of adenylic acid with (2'-5')-phosphodiester linkages and a triphosphate at the 5'-end; (2'-5')-ppp3'dA(p3'dA).sub.n, oligomer of 3'-deoxyadenylic acid with (2'-5')-phosphodiester linkages and a triphosphate at the 5'-end; (2'-5')A(pA).sub.n or core oligomer, oligomer of adenylic acid with (2'-5')-phosphodiester linkages; (2'-5')A(pA).sub.2 or core trimer, adenylyl(2'-5')adenylyl(2'-5')adenosine; (2'-5')A(pA).sub.3 or core tetramer, adenylyl(2'-5') adenylyl(2'-5')adenylyl (2'-5')adenosine; (2'-5')3' dA(p3'dA).sub.2 or core trimer analog, 3'-deoxyadenylyl (2'-5')3'-deoxyadenylyl(2'-5')3'-deoxyadenosine; poly(rI).poly(rC)agarose, poly(inosinate).poly(cytidylate) double stranded polynucleotide covalently bound to agarose though poly(rI); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; A, adenosine; 3'dA, 3'-deoxyadenosine (3'-deoxyadenylate); pA, 5'-AMP; p3'dA, 3'-deoxy-5'-AMP or 3'-deoxyadenylate; 3'-dATP, 3'-deoxyATP; BAP, bacterial alkaline phosphatase; SVPD, snake venom phosphodiesterase I; ATP; adenosine triphosphate; AMP, adenosine monophosphate.
Under ordinary circumstances a cell will contain ATP and ATP synthetase and an endonuclease which will not interact. In a manner not entirely understood where viral infection of the cell is about to occur interferon reacts upon the cell causing the synthetase to act upon the ATP to form (2'-5')oligo(A) having the general designation (2'-5')p.sub.m A(pA).sub.n where m is 1,2 or 3 and n may be 1-14, preferably 1-5. Upon intrusion of the virus which contains double stranded ribonucleic acid, the (2'-5') oligo (A) activates the endonuclease which then destroys the messenger ribonucleic acid of the virus, preventing replication of the virus. In this anti-viral defense mechanism of the cell, generally speaking, the (2'-5') oligo (A) produced by the cell, is destroyed. The half life of this oligomer is extremely short and cannot therefore be utilized as an externally administered anti viral material without being immediately destroyed by the cell defense mechanisms. It would be desirable therefore to provide an endonuclease activator of substantially greater half life than the (2'-5') oligo (A) which can be administered as a anti-viral material without being immediately destroyed by the cellular defense mechanisms.
The existance of the (2'-5') phosphodiester bond in nature is an item of comparatively recent knowledge (Cory et al Biochim Biophys Acta 103, 646 (1965)).
In classical work by Lord Todd it was shown that in the more commonly recognized (3'-5') phosphodiester bond compound a cylic linkage between the 3'-hydroxyl group and the bridging phosphate group between the two ribose moieties was essential for the hydrolytic cleavage of the phosphodiester linkage. It is further known that all reactions involving adenosine triphosphate depend upon the presence of an hydroxyl moiety either in the 2'- or in the 3'-position of the ribose nucleus of the adenosine moiety. In considering the reasons for the rapid destruction of (2'-5') oligo (A) in the cell it will be seen that the 2'- position can not participate since it does not bear a hydroxyl group.
The inventors herein therefore postulated that since the 2'-position was not available for reaction, the degradation of the oligomer might proceed via a cyclic mechanism involving the 3'-hydroxyl in a manner similar to the involvement of the 2'-hydroxyl as shown by Todd. The inventors herein therefore further postulated that the absence of a hydroxyl moiety at the 3'-position might interupt the hydrolytic degradation of the (2'-5') phosphodiester linkage and thus lead to an increased half life for the resulting product. They further postulated that if the presence of the aforesaid 3'-hydroxyl group was not essential for the activation of the endonuclease there might be provided a material which would carry out the endonuclease activating function of (2'-5') oligo (A) while having a substantial greater half-life.
Primary exposure to Epstein Barr virus (EBV) in childhood usually results in a silent infection. If infection with EBV is postponed until adolescence, a more serious illness, infectious mononucleosis, develops in approximately 40 percent of the individuals. Although usually throught of as resulting in a self-limiting lymphoproliferative disease, it is becoming increasingly clear that primary infection with EBV can result in life-threatening illness in both adolescents and children. This fact adds support that the morbidity and mortality associated with EBV infection is more serious than originally thought. EBV infection has also been associated with several malignancies, Burkitt's lymphoma and nasopharyngeal carcinoma and Hodgkin's disease.
In vitro systems have been used to evaluate the ability of compounds with antiviral properties to inhibit EBV replication, including 1-.beta.-D-arabinofuranosylthymine and 9-.beta.-D-arabinofuranosyladenine. One of the difficulties encountered with nucleoside analogs as antiviral agents is specificity for the virus-infected cell and concomitant cytotoxicity. Another difficulty with nucleoside analogs (such as 9-(2-hydroxyethoxymethyl)guanine and 5'-amino-5'-deoxyiodouridine) as antiviral agents is that they can act only after the mammalian cell is infected by a virus and the viral deoxythymidine kinase is expressed. Because of these difficulties with the use of nucleosides, interest has grown in the use of autochthonous interferon as an antiviral and antineoplastic agent. However, the development and use of interferon as an antiviral agent has been limited due to scarcity and problems associated with delivery to target cells. Another disadvantage of interferon as an antiviral/antitumor agent is the recent report of the formation of antibodies to interferon. Few in depth in vitro studies have been reported on the effects of purified interferon on EBV replication in human lymphocytes, although fibroblast transformation by oncogenic DNA viruses has been successfully inhibited by exogenously added interferon. It has recently reported that the transformation of adult, but not newborn, lymphocytes by EBV and phytohemagglutinin is inhibited by interferon.
Interferon-treated cells develop the antiviral state by the induction of (2'-5')(A).sub.n synthetase and a protein kinase. The (2'-5')A(pA).sub.n formed by the synthetase in the presence of double stranded RNA activates a latent endoribonuclease which degrades messenger RNA. Thus it appeared to the inventors herein that Epstein Barr virus would provide an interesting test model for the aforementioned hypothesis.